Explain how Illumina NGS sequencing data is generated
How to evaluating NGS data [Practical]
Introduction to DNA
What is DNA?
Deoxyribonucleic acid (/diːˈɒksɪˌraɪboʊnjuːˌkliːɪk, -ˌkleɪ-/ (DNA) is a molecule composed of two polynucleotide chains that coil around each other to form a double helix carrying genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. - Wikipedia
Calculates a probability the ‘base call’ is correct i.e. blurry or weak image?
Note the index in the sequence (sample-specific barcode)
Group each recorded sequence or ‘reads’ with those with the same index
a.k.a. demultiplexing
FASTQ File
FASTQ format is a text-based format for storing both a biological sequence (usually nucleotide sequence) and its corresponding quality scores. Both the sequence letter and quality score are each encoded with a single ASCII character for brevity. - Wikipedia
@ <read id, e.g. machine ID, location on flowcell> <extra metadata>
<DNA sequence; Note: N = base couldn't be called!>
+ <a separator>
<base quality scores for each nucleotide in sequence>
a.k.a. sequencing ‘artefacts’ not the real DNA molecule!
Base qualities
Cycle-quality decay
Paired-end sequencing!
Merge together: better confidence in base call
Recap
DNA molecules essentially:
Made up of nucleotides (ACTG)
Two strands: complementary base pairs (C-G, A-T)
aDNA are fragmented molecules: short
NGS Sequencing:
Massively multiplexed: millions DNA molecules at once
Add adapters to bind to a glass slide
Make new strand, adding florescent nucleotides
Fire laser at each nucleotide and take photo
Results in **FASTQ* file
Has base quality scores
Practical: Introduction to NGS data processing
Working on the command-line
What is the command line?
A command-line interface (CLI) processes commands to a computer program in the form of lines of text. - Wikipedia
i.e. use words, not point and click with mouse
Important: more efficient/scalable & more reproducible
Most bioinformatics work is performed via command line
Often as working on remote servers (i.e. very large computers with no screen)
Logging into a server
Open browser
Go to:
Log-in with your credentials
The command line
A command prompt (or just prompt) is a sequence of (one or more) characters used in a command-line interface to indicate readiness to accept commands. - Wikipedia
james_fellows_yates@bionc21:~$
<username>@<machine_name>:<current_directory>$
Everything after$ is where you type your command
Never copy and paste the prompt!
⚠️Prompts look different on different machines!
Your first command
Type in everything after the prompt, and press enter/return (⏎) on your keyboard with
$ echo "Hello world!"
Hello world!
Command typically consists of:
Program/software/tool name
Arguments (e.g. input files)
Options or flags (e.g. -h or --help)
Move around
What is in the room (directory)
$ ls
Lets go in the directory, and see what’s in there!
nf-core/eager is a scalable and reproducible bioinformatics best-practise processing pipeline for genomic NGS sequencing data, with a focus on ancient DNA (aDNA) data. It is ideal for the (palaeo)genomic analysis of humans, animals, plants, microbes and even microbiomes.
Pipeline (software): a chain of data-processing processes or other software entities
@jfy133
I, Madprime, CC0, via Wikimedia Commons
Estevezj, CC BY-SA 3.0 via Wikimedia Commons
Konrad Förstner, CC0, via Wikimedia Commons
Bronner et al. (2013) Current Protocols in Human Genetics, DOI: (10.1002/0471142905.hg1802s79)
DMLapato, CC BY-SA 4.0, via Wikimedia Commons
Abizar Lakdawalla , CC BY 3.0, via https://openlab.citytech.cuny.edu/
EMBL-EBI Training, CC BY-SA 4.0, via https://www.ebi.ac.uk/training/